Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt
Abstract Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test it...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-02-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-021-01529-y |
| _version_ | 1818661802434625536 |
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| author | Claudia Durall Kateryna Kukil Jeffrey A. Hawkes Alessia Albergati Peter Lindblad Pia Lindberg |
| author_facet | Claudia Durall Kateryna Kukil Jeffrey A. Hawkes Alessia Albergati Peter Lindblad Pia Lindberg |
| author_sort | Claudia Durall |
| collection | DOAJ |
| description | Abstract Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle. |
| first_indexed | 2024-12-17T04:50:51Z |
| format | Article |
| id | doaj.art-53adcb7064424cddbe147dd1d0543c5f |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-17T04:50:51Z |
| publishDate | 2021-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-53adcb7064424cddbe147dd1d0543c5f2022-12-21T22:02:54ZengBMCMicrobial Cell Factories1475-28592021-02-0120111410.1186/s12934-021-01529-yProduction of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shuntClaudia Durall0Kateryna Kukil1Jeffrey A. Hawkes2Alessia Albergati3Peter Lindblad4Pia Lindberg5Microbial Chemistry, Department of Chemistry-Ångström, Uppsala UniversityMicrobial Chemistry, Department of Chemistry-Ångström, Uppsala UniversityAnalytical Chemistry, Department of Chemistry-BMC, Uppsala UniversityMicrobial Chemistry, Department of Chemistry-Ångström, Uppsala UniversityMicrobial Chemistry, Department of Chemistry-Ångström, Uppsala UniversityMicrobial Chemistry, Department of Chemistry-Ångström, Uppsala UniversityAbstract Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.https://doi.org/10.1186/s12934-021-01529-ySynechocystis PCC 6803TCA cycleGlyoxylate shuntSuccinatePhosphoenolpyruvate carboxylaseSuccinate dehydrogenase |
| spellingShingle | Claudia Durall Kateryna Kukil Jeffrey A. Hawkes Alessia Albergati Peter Lindblad Pia Lindberg Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt Microbial Cell Factories Synechocystis PCC 6803 TCA cycle Glyoxylate shunt Succinate Phosphoenolpyruvate carboxylase Succinate dehydrogenase |
| title | Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt |
| title_full | Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt |
| title_fullStr | Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt |
| title_full_unstemmed | Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt |
| title_short | Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt |
| title_sort | production of succinate by engineered strains of synechocystis pcc 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt |
| topic | Synechocystis PCC 6803 TCA cycle Glyoxylate shunt Succinate Phosphoenolpyruvate carboxylase Succinate dehydrogenase |
| url | https://doi.org/10.1186/s12934-021-01529-y |
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